Most tissue engineering techniques basically consist of seeding a tissue scaffold or culture dish with cells that are grown in an incubator. The scaffold fabrication and the cell seeding are two separate processes. These techniques are very limited in their level of sophistication. The scaffolds tend to be simple structures made out of a single material, with some post-processing techniques in the slightly more complicated scaffolds. Organized, heterogeneous cellular structures are very difficult to create, and impossible to create at the complexity level of an organ using standard techniques. Seeding these kinds of scaffolds may not be enough to stimulate the cells into responding in the desired manner. A complex scaffold is required to elicit complex behavior from a cell. A new generation of tissue scaffolds is required to take the next step in tissue engineering which essentially moves away from simple scaffolds toward complex scaffolds. A cell is a very sophisticated machine with programming built into its genetic code. A complex scaffold takes advantage of this built-in programming through the incorporation of various biological factors that direct cell growth, migration, differentiation, and expression. In addition, constructs can be created that could help with the flow and transport of vital nutrients and oxygen, and the removal of waste products.
Layered manufacturing has been suggested as being well suited to the field of biology. This has resulted in much research being conducted within the field of computer-aided tissue engineering (CATE). Unfortunately, many Solid Freeform Fabrication (SFF) techniques are not biologically friendly using techniques that cannot handle a wide range of wet materials, gels or solutions. Also, many SFF techniques utilize harsh solvents, high temperatures, high pressures, and other factors that are not conducive to biological systems. Many SFF techniques, such as stereolithography, fused deposition methods, and powder/binder-based techniques, are capable of creating tissue scaffolds, but cannot directly deposit cells or biological factors into the scaffold. This has resulted in the creation of different techniques to handle direct cell deposition.
Weiss, et al. have described a method for building bone tissue scaffolds using SFF (Reischmann et al. Electrotechnik und Informationstechnik 2002 7/8:248-252; Weiss et al. Journal of Manufacturing Systems 1997 16(4): 239-248). This process consists of taking a CAD model of a three-dimensional structure of a bone implant, slicing the model into layers, taking laminated sheets of scaffold material, seeding the layers with cells or growth factors, and stacking them on top of each other. This process was designed for the purpose of constructing bone implants, not to provide a flexible process of creating various types of organs or biologically/chemically integrated systems and thus has several disadvantages with respect to construction of tissue engineering devices. For example, the method is limited in materials since soft, gel-like materials cannot be used as scaffold layers. This is a problem since many biological parts are soft or wet. Also, each layer of the scaffold is made with one type of sheet material. Thus, it is difficult to have two or more different materials within the same layer level. Accuracy and recalibration is an issue as well since the scaffold layers are moved from station to station. Thus, although a simple scaffold can be created by this method, a complex scaffold with controlled concentration gradients is difficult, if not impossible, to create. This is a serious disadvantage since cells are very responsive to even the slightest differences in concentration gradients.
Yan and Xiong, et al. have disclosed the concept of using layered manufacturing methods and multi-nozzle deposition extrusion and jetting processes (Xiong et al. Scripta Materialia 2002 46:771-776; Yan et al. Materials Letters 2003 57:2623-2628). Their process includes spraying and deposition of heterogeneous materials with different material properties. However, full CAD integration is not described. Nor is there any description of the ability to import an assembly of multiple STL files for printing a complex, heterogeneous, three-dimensional structure. This is a vital design component when building complex parts such as biomimetic parts where MRI or CT data is incorporated into the final design, or integrating both biomimetic parts and non-biomimetic parts into a novel scaffold design.
A SFF method using a syringe-based system to dispense liquids, which is well suited for working with biological materials such as cells and hydrogels has also been described (Landers et al. Macromol Mater Eng 2000 282:17-21; Landers et al. Kunststoffe/plast Europe 2001 91(12): 21-23; Landers et al. Biomaterials 2002 23:4437-4447). The primary focus of this method is the building of scaffolds and seeding the scaffold. The deposition system used is a single nozzle device that requires cartridge swapping to change materials. This is not a very practical system for depositing multiple, heterogeneous materials such as different types of cells and growth factors all within the same scaffold layer. Further, it is difficult to take a multiple part assembly of STL files and print out a complex, biologically designed scaffold utilizing this method. Thus, there are limitations in this method with respect to the CAD integration aspect as well.
A syringe-based system for the extrusion of hybrid polymer materials embedded with glass using layered SFF manufacturing has also been described (Calvert et al. Materials Science and Engineering 1998 C6:167-174). This system also uses a single nozzle and does not incorporate CAD, thus being limited to simple designs written in Microsoft Qbasic. This system is not capable of creating heterogeneous designs within a single layer. Thus, this system is sufficient for creating basic scaffolds, but falls short of being able to create intricate scaffolds containing both biomimetic and non-biomimetic features.
A microsyringe deposition system has also been described (Vozzi et al. Materials Science and Engineering 2002 C20:43-47; Vozzi et al. Biomaterials 2003 24:2533-2540). This system utilizes a single-nozzle deposition system which has fine resolution, but is limited because of the glass capillary used for deposition. The glass capillary limits the range of viscosities that are usable due to pressure limits, and also limits the types of solutions and suspensions that can be deposited due to clogging. The device is envisaged for integration with CAD, but whether their working device could actually utilize STL files is unclear. Also, the single nozzle system makes multi-material, heterogeneous deposition difficult.
A single-nozzle, automated extrusion system that can utilize basic STL files has been described as well (Ang et al. Materials Science and Engineering 2002; C20:35-42). It is unclear whether this system can be utilized to produce multi-part, heterogeneous STL files. This single nozzle process also makes constructing complex parts very difficult, and limits the diverse range of materials available for deposition.
Mironov, et al. discuss the basic principles of organ printing, which involves direct deposition of cells using a multi-nozzle printing system (Mironov et al. TRENDS in Biotechnology 2003 21(4):157-161). A general basic concept of organ printing involving CAD in the preprocessing stage incorporating either patient specific MRI/CT data or artificial computer generated biomimetic constructs is set forth. However, there is no mention of the value of CAD beyond simply imitating biology. In addition, there are serious limitations with their disclosed multi-nozzle system which uses the same type of syringe thus limiting the types of materials that can be deposited. In order to build good 3-dimensional structures, relatively viscous solutions are required, which means high pressure. High pressure, however, may not be compatible with cells. High pressure systems handling viscous materials have the problem of not being able to deposit fine structures with fine concentration gradients. Finally, there is a flaw in the process described in this reference because they do not consider the fact that CAD programs do not have heterogeneous material capabilities. Thus, they neglect a non-trivial and difficult step by assuming that they can create a multi-material part in CAD and print it out using multiple nozzles, which is not necessarily the case.
U.S. Pat. No. 6,139,574 (Vacanti, et al. Oct. 31, 2000) discloses vascularized tissue regeneration matrices formed by SFF techniques. Use of CAD and SFF techniques for the creation of tissue scaffolds is mentioned. Further, they mention the possibility of using multiple printheads and different kinds of SFF techniques. However, there is no description of direct cell deposition. The reason for this is that the method described is not biologically friendly to cells. Thus, the described method requires depositing the scaffold material and bioactive materials first to create the scaffold, and then seeding the cells externally relying upon cell migration to populate the scaffold. Further, the inkjet printing method described by Vacanti creates problems for cellular deposition unless significant steps are taken to protect cells from shear stresses that would tear the cell apart.
U.S. Pat. No. 6,143,293 (Weiss, et al. Nov. 7, 2000) discloses assembled scaffolds for three dimensional cell culturing and tissue generation. The method used is primarily oriented towards building hard, bone-type scaffold structures and creation of soft, gel-like scaffolds using this method may be difficult. Further heterogeneous capabilities are limited to materials that can be added on top of the layer, but not within the layer itself. The method of Weiss et al. also utilizes prefabricated layers thus necessitating an assembly stage, which then requires extra steps to calibrate, align, and affix the layers. Means for affixing the layers such as barbs, or other mechanical affixing means is a disadvantage that may result in later complications due to wear, bone remodeling, or incompatibilities in material properties. The method described by Weiss et al. thus lacks versatility and flexibility.
U.S. Pat. No. 6,027,744 and U.S. Pat. No. 6,171,610 (Vacanti, et al. Feb. 22, 2000 and Vacanti, et al. Jan. 9, 2001) describe guided development and support of hydrogel-cell compositions. Methods described therein use hydrogel-cell compositions as a means of tissue scaffold construction and rely upon injecting the hydrogel-cell material into the tissue scaffold. The described method does not include layered fabrication methods or CAD. Direct deposition of cells into a scaffold while constructing the scaffold is also not mentioned.
U.S. Pat. No. 6,176,874 (Vacanti, et al. Jan. 23, 2001) discloses vascularized tissue regeneration matrices formed by SFF fabrication techniques. Again, the described method does not include layered fabrication methods or CAD nor direct deposition of cells into a scaffold while constructing the scaffold.
U.S. Pat. No. 6,454,811 (Sherwood, et al. Sep. 24, 2002) discloses composites for tissue regeneration and methods of manufacture thereof. This method primarily focuses on three-dimensional printing (3DP) for tissue engineering. Although there is mention that other methods of SFF could be used, no explicit details are provided. Further, there is no mention of CAD integration, heterogeneous materials, multi-part assemblies, and multi-nozzle printing within a CAD environment. In addition, the majority of the SFF methods described are not biologically friendly for direct cell deposition. For example, stereo-lithography, selective laser sintering, and fused deposition modeling cannot directly deposit cells due to heating and toxicity issues which will kill cells. Ballistic particle manufacturing also has problems due to shear stresses that can damage cells, which are very sensitive and require low pressure or a protective method to reduce the shear stresses experienced by the cell. The described 3DP method is also unable to directly seed cells into the interior of the part that is being constructed. This process also requires post-processing in which powder, which functions both as the part and the support material, has to be removed after finishing the printing process. Thus, while this method can be used to create porous structures, the pores are filled with powder during the printing stage. It is only after printing has been completed that the powder is removed to open up the pores. Thus, cells cannot be directly printed at specific locations inside the part. Instead, cells must migrate from the outside of the scaffold, into the interior of the scaffold. This is a serious disadvantage when trying to create reproducibility between histotypic or organ culture samples. Finer features require additional post-processing, such as salt-leaching, which again makes direct cellular deposition impossible.
U.S. Pat. No. 6,547,994 (Monkhouse, et al. Apr. 15, 2003) describes a process for rapid prototyping and manufacturing of primarily drug delivery systems with multiple gradients, primarily involving a 3DP technique. These 3DP techniques share the same shortcomings as described for U.S. Pat. No. 6,454,811.
U.S. Pat. No. 6,623,687 (Gervasi, et al. Sep. 23, 2003) describes a process for producing three-dimensional objects by constructing an interlaced lattice construct using SFF to create a functional gradient material. There is brief mention of the possibility of using this technique to create tissue engineered constructs such as veins and arteries. However, there is no demonstration of use in this application.